KR20180058829A - How to Treat Mesothelioma with EZH2 Inhibitor - Google Patents

Abstract

The present disclosure provides a method of treating a hydroblastoma in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an enhancer (EZH2) inhibitor of esthetic estrogen 2. In a preferred embodiment of this method, the subject is a pediatrician and the EZH2 inhibitor is thymometast.

Description

How to Treat Mesothelioma with EZH2 Inhibitor

Related application

This application claims priority to U.S. Provisional Application No. 62 / 238,074 (Oct. 6, 2015) and No. 62 / 299,312 (Feb. 24, 2016), each of which is incorporated herein by reference in its entirety. Claim the benefit.

The field of the present disclosure

This disclosure relates to the field of small molecule therapy, cancer, and methods of treatment of the rare cancer types.

The need for effective treatments for certain cancers caused by loss of function or genetic modification of subunits in the SWI / SNF chromatin remodeling complex resulting in EZH2-dependent carcinogenesis has long been and is not being met.

The present disclosure provides a method of treating a hydroblastoma in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an enhancer (EZH2) inhibitor of esthetic estrogen 2. The method of treatment of hydroblastoma of the present disclosure may include preventing and / or inhibiting the proliferation of hydroblastoma cells.

In certain embodiments of the methods of the disclosure, the EZH2 inhibitor is

(Compound A or thymometastate), or a pharmaceutically acceptable salt thereof.

In certain embodiments of the methods of the disclosure, the EZH2 inhibitor is

Their stereoisomers, their pharmaceutically acceptable salts and / or solvates.

In certain embodiments of the methods of the disclosure, the EZH2 inhibitor is

Or a pharmaceutically acceptable salt thereof.

In certain embodiments of the methods of the disclosure, the EZH2 inhibitor is

,

,

Stereoisomers, pharmaceutically acceptable salts and / or solvates thereof.

In certain embodiments of the methods of the disclosure, the EZH2 inhibitor is

,

,

Stereoisomers, pharmaceutically acceptable salts and / or solvates thereof.

In certain embodiments of the methods of the disclosure, the EZH2 inhibitor is

Their stereoisomers, their pharmaceutically acceptable salts and / or solvates.

The EZH2 inhibitor of the present disclosure can be administered orally. For example, EZH2 inhibitors may be formulated as oral tablets or suspensions.

EZH2 inhibitors of this disclosure may be formulated for administration by any route to CSF (CSF). Exemplary routes of administration to CSF include, but are not limited to, intra-spinal, intracranial, intraspinal, or intranasal routes.

In certain embodiments of the methods of the disclosure, including, but not limited to, embodiments wherein the EZH2 inhibitor is formulated into an oral tablet, the EZH2 inhibitor of the present disclosure is administered at a dose of from 10 mg / kg / day to 1600 mg / kg / Lt; / RTI > per day. The EZH2 inhibitor of the present disclosure may be administered at a dose of about 100 mg, about 200 mg, about 400 mg, about 800 mg, or about 1600 mg. The EZH2 inhibitor of the present disclosure may be administered at a dose of about 800 mg. EZH2 inhibitors of this disclosure may be administered once or twice a day (BID). For example, the EZH2 inhibitor of the present disclosure can be administered BID at a dose of 10 mg / kg / day to 1600 mg / kg / day. The EZH2 inhibitor of the present disclosure may be administered BID at a dose of 800 mg.

In certain embodiments of the methods of this disclosure, including, but not limited to, embodiments wherein an EZH2 inhibitor is formulated to be formulated into an oral suspension and / or administered by CSF by any route, the EZH2 The inhibitor may be selected from the group consisting of 50%, 60%, 70%, 80%, 90% or any of the values of the steady state plasma and / or CSF concentration under the curve area (AUC) (AUC _SS ) of the EZH2 inhibitor , Wherein AUC _SS is determined after BZID administration of an EZH2 inhibitor to an adult subject at a dose of 10 mg / kg / day to 1600 mg / kg / day.

In certain embodiments of the methods of this disclosure, including, but not limited to, embodiments wherein an EZH2 inhibitor is formulated to be formulated into an oral suspension and / or administered by CSF by any route, the EZH2 The inhibitor may be administered at a dose of 230 mg / m 2 to 600 mg / m 2 including the end point. The EZH2 inhibitor of the present disclosure can be administered in a dose of 300 mg / m 2 to 600 mg / m 2. The EZH2 inhibitor of the present disclosure can be administered in a dose of from 230 mg / m 2 to 305 mg / m 2 including the endpoint. The EZH2 inhibitor of the present disclosure can be administered in a dose of 240 mg / m < 2 >. The EZH2 inhibitor of the present disclosure can be administered at a dose of 300 mg / m < 2 >. The EZH2 inhibitor of the present disclosure may be administered once or twice a day (BID). For example, the EZH inhibitor of the present disclosure may be administered BID at a dose of from about 230 mg / m 2 to about 600 mg / m 2, including the endpoint.

For example, the EZH2 inhibitor of the present disclosure can be administered at a dose of about 60% of the area under the curve (AUC) under normal conditions (ACU _SS ) after 1600 mg administered twice a day to adult subjects have. Thus, the EZH2 inhibitor of the present disclosure, administered at a dose of about 60% of the area under the curve (AUC) in the steady state (ACU _SS ) after administration of 1600 mg twice a day to adult subjects, 600 mg / m 2 or at a dose of at least 600 mg / m 2 per day. In certain aspects of these examples, the subject to be treated with the EZH2 inhibitor is a pediatric subject.

For example, the EZH2 inhibitor of this disclosure may be administered at a dose of about 80% of the area under the curve (AUC) in the steady state (AUC) (ACU _SS ) after 800 mg administered twice a day to an adult subject . Therefore, the EZH2 inhibitor of the present disclosure administered at a dose of about 80% of the area under the curve (AUC) in the steady state (ACU _SS ) after 800 mg administered twice a day to adult subjects is about 390 Mg / m 2 or at least 390 mg / m 2 per day. In certain aspects of these examples, the subject to be treated with the EZH2 inhibitor is a pediatric subject.

The subjects of this disclosure may be pediatric subjects. For example, the pediatric subject of the present disclosure may be between 6 months and 21 years of age, including the age of the endpoint. The pediatric subject of the present disclosure may be 1 to 18 years of age, including the age of the endpoint. The pediatric subjects of this disclosure may be less than 10 years of age. The pediatric subjects of this disclosure may be 5 years of age or younger. The pediatric subject of the present disclosure may be six months to one year of age, including the age of the endpoint.

The disclosure provides a method of treating a hydroblastoma in a subject in need of treatment of the hydroblastoma, comprising administering to the subject a therapeutically effective amount of a tadalometastate, wherein the therapeutically effective amount is 1 day (BID) of at least 300 mg / m 2, and subjects are between 6 months and 21 years of age, including the end point.

Figures 1a and 1b are a series of western blot analysis of cell lines (RD and SJCRH30) with wild type SNF5 and cell lines with mutant SNF5.
2a to 2e show that the SNF5 mutant cell line A204 (FIG. 2c), G401 (FIG. 2d) and G402 (FIG. 2e) are different from the wild type strain RD ). ≪ / RTI >
3A-3D are a series of bar graphs showing that the G401 SNF mutant cell line, after 7 days in soft agar, responds to compound D compared to the wild type cell line RD. Figure 3a shows cell line RD (5,000 cells / well). Figure 3b shows G401 cells (5,000 cells / well). Figure 3c shows G401 cells in 2D growth. Figure 3d shows G401 cells (10,000 cells / well).
Figures 4a-4d are four graphs showing that the G401 SNF5 mutant cell line is susceptible to Compound A in vitro. The wild type cell lines SJCRH30 (Fig. 4A) and RD (Fig. 4C) and the SNF5 mutant cell line G401 (Fig. 4B) and A204 (Fig. 4D) were pretreated with the indicated concentrations of Compound A for 7 days and re-referenced on day 0. The cell viability was determined by the CellTiter-Glo ^® luminescent cell viability assay.
Figures 5a-5e are a series of graphs showing the continuous regression of the G401 xenograft (malignant hepatocellular tumor model) in Compound A treatment. Figure 5a: Tumor regression caused by a given dose of Compound A. Figure 5b: Tumor regression caused by twice daily dosing of a given dose of Compound A. The data represent the mean value ± SEM (n = 8). Compound administration was discontinued on day 28. Figure 5c: EZH2 target inhibition in G401 xenograft tumor tissues collected from a parallel cohort of mice on day 21. Each point represents the ratio of H3K27Me3 to total H3. Horizontal lines represent group mean values. BLLQ = Below the lower limit of the specified amount. Figures 5d and 5e: Immunohistochemical staining results of tumor histone methylation of tumor samples from vehicle treated mice (Figure 5d) and Compound A treated (125mg / kg) mice (Figure 5e).
Figure 6 is a graph showing the location of ATRX mutations identified in SCLC cell lines.
FIG. 7A is a graph showing that LNCAP prostate cancer cells inhibit cell growth in a dose-dependent manner during in vitro treatment of Compound D. FIG.
Figure 7b is a graph showing the IC50 values of compound D against WSU-DLCL2 cells and LNCAP cells at days 11 and 14.
8A-8C are three graphs showing that ATRX mutant SCLC strains NCI-H446 (FIG. 8A), SW1271 (FIG. 8B) and NCI-H841 (FIG.
Figures 9a-9c show that SCLC strain NCI-H841 after treatment with vehicle (Figure 9a) or with concentration 4.1E-02 uM of compound D (Figure 9b) or 3.3 uM of concentration of compound D (Figure 9c) Three microscope images.
Figures 10a-10c are a series of graphs showing the effect of Compound A on intracellular universal histone methylation and cell viability. 10a: Chemical structure of Compound A (or thymometast). 10b: concentration-dependent inhibition of intracellular H3K27Me3 levels in G401 and RD cells. Figure 10c: In vitro selective inhibition of SMARCB1 deletion G401 cell proliferation by Compound A (as measured by ATP content). On day 7, G401 cells (panels a and b) and RD cells (panels c and d) were re-assayed at their original inoculum density. Each point represents the mean (n = 3) for each concentration.
11A and 11B are a series of graphs depicting biochemical mechanisms of functional studies. The IC ₅₀ values of Compound A are increased as the SAM concentration increases (FIG. 11 a) and minimally affected by the increase in oligonucleosomal concentration (FIG. 11 b), indicating that the competitive mechanism of SAM and the ratio of the nucleosomes Indicating a competitive mechanism.
12A and 12B are a series of panels demonstrating the basis for expression of SMARCB1 and EZH2 in the cell line and the specificity of Compound A for inhibition of intracellular histone methylation. Figure 12a: Cell lysates were analyzed by immunoblotting using antibodies specific for SMARCB1, EZH2 and actin (loading control). Figure 12b: Selective inhibition of G401 and RD cell intracellular H3K27 methylation. Cells were incubated with Compound A for 4 days, and histone extracted with acid was analyzed by immunoblotting.
Figures 13a and 13b are a series of bar graphs demonstrating that Compound A induces G ₁ arrest and apoptosis in SMARCBl deficient MRT cells. Cell cycle analysis (by flow cytometry) and cell apoptosis determination results (TUNEL) in RD (panel A) or G401 cells (panel B) during incubation with either vehicle or 1 μM of compound A for periods of up to 14 days By black). G ₁ arrest was observed at 7 days, and apoptosis was induced at 11 days. The data are expressed as mean values ± SEM (n = 2). The indicated DMSO control values are mean ± SEM at each time point. The cells were split and re-seeded at the original inoculum density on days 4, 7, and 11.
14A to 14C are a series of graphs showing that Compound A induces the expression of SMARCB1 regulatory genes and changes in cell morphology. Figure 14a: Basal expression of SMARCB1 regulated gene (measured by qPCR, n = 2) in G401 SMARCB1 -deficient cells compared to RD control cells. 14b: G401 and RD cells were incubated with either DMSO or 1 [mu] M Compound A for 2 days, 4 days and 7 days. Gene expression was determined by qPCR (n = 2) and expressed relative to the DMSO control at each time point. The panels a to j correspond to the genes GLI1, PTCh1, DOCK4, CD133, PTPRK, BIN1, CDKN1A, CDKN2A, EZH2, and MYC, respectively. Figure 14c: G402 cells were incubated for 14 days with either DMSO (left panel) or 1 μM Compound A (right panel). Cells were split and re-seeded at the original inoculum density on day 7.
Figures 15a-15d are a series of graphs showing body weight, tumor regression and plasma levels in G401 xenografted mice treated with Compound A; 15a: The body weights of animals in which Compound A was treated for 28 days on a BID schedule were measured twice a week. Data are presented as means ± SEM (n = 16 for 21 days, n = 8 for days 22 to 60). Figure 15b: Tumor degeneration (mean value ± SEM, n = 16; * p <0.05, ** p <0.01, repeated measures ANOVA, Dunnett post-test vs. vehicle). 15c: tumor weight of 8 mice euthanized at 21 days (**** p < 0.0001, Fisher exact test). 15d: Plasma was collected 5 days before and 3 hours after administration of Compound A on day 21, and compound levels were determined by LC-MS / MS. On day 21 animals were euthanized and tumors were collected 3 hours after dosing. Tumor homogenates were generated and LC-MS / MS analysis was performed to determine the concentration of Compound A. Note that tumor compound levels could not be determined from all animals, especially from high-dose groups of animals, because the xenografts on day 21 were too small. The dots represent values for individual animals; Horizontal lines represent group mean values.
Figures 16a-16c are a series of graphs showing that Compound A abrogates SMARCB1 -deficient MRT xenografts in SCID mice. Figure 16a: Tumor regression induced by administration of compound A twice daily (BID) at the indicated dose for 28 days. Compound administration was discontinued on day 28, and tumors were allowed to regrow until reaching 2000 ((data are shown as mean values ± SEM (n = 8)). 16b: EZH2 target inhibition in G401 xenograft tumor tissues collected from mice euthanized at 21 days. Each point shows the ratio of H3K27Me3 to total H3, as measured by ELISA. Horizontal lines represent group mean values; The gray symbols are values outside the ELISA standard curve. 16c: Gene expression changes in G401 xenograft tumor tissues collected from mice treated with Compound A for 21 days. The panels a to d correspond to the genes CD133, PTPRK, DOCK4 and GLI1, respectively. The data are presented as a multiple of the vehicle variation ± SEM (n = 6 for the n = 6, 500 mg / kg group) (* p <0.05, ** p <0.01, **** p <0.0001, vs Vehicle, Fisher exact assay).
17 is a schematic diagram showing the posterior control of gene expression. The combination of histone transformations encodes information that controls developmental cell identification and fate determination, as well as integrated activation or suppression of genetic programs.
18 is a graph showing that EZH2 is overexpressed in a hydroblastoma and is associated with chromosome 7 amplification. The hollowed rod represents a balanced chromosome 7, while the rod with a thin parallelogram represents chromosome gain 7.
19 is a schematic diagram showing the control of histone lysine methylation by EZH2 and MLL.
FIG. 20A is a graph showing the overall survival (OS) probability as a function of elapsed time (months) since the diagnosis of male blastoma. Histone lysine methylation is altered in hydroblastoma. The abundance ratio of H3K27me3 is increased in hydroblastoma cells as compared with control cells.
FIG. 20B is a graph showing the overall survival (OS) probability as a function of the elapsed time (months) since the diagnosis of a male blastoma. Histone lysine methylation is altered in hydroblastoma. The abundance ratio of H3K27me3 is increased in hydroblastoma cells as compared with control cells.
21A is a series of photographs and graphs showing the abundance ratio of H3K27me3 and H3K27Me3 in the squamous cell line. The data demonstrate the deregulation of histone secretion in the hydroblastoma.
Figure 21B is a graph showing overall survival (OS) probability as a function of elapsed time (months) since the diagnosis of a bladder cell tumor for a subject with a dystrophic histone methylation at H3K27me3 and / or H3K27Me3.
Figure 22A is a graph demonstrating that inhibition of EZH2 by short hairpin EZH2 (shEZH2) constructs inhibits vesicular blast cell growth (growth of DAOY blastoma cell line) compared to negative control constructions.
Figure 22b shows a series of demonstrations that inhibition of EZH2 by short hairpin EZH2 (shEZH2) construct inhibits vesicular blast cell growth (DAOY male blastoma cell line growth), as compared to negative control and / or empty pSIF vector control &Quot;
23A is a schematic illustration showing the mechanism by which a carcinogenic dependence on EZH2 occurs by loss of INI1 in a tumor.
23B is a graph showing the tumor-free survival percentage of INI1-deficient mice as a function of time (days) when EZH2 is knocked out. EZH2 knockout reverses the carcinogenesis induced by INI1 loss.
Figure 24a is a series of photographs showing control or EZH2 inhibitor treated (DNZep treated) anomalous teratocarcinoma (ATRT) at 1, 3, 5 and 7 days post-treatment. Inhibition of EZH2 inhibits ATRT cell self-renewal.
FIG. 24B is a graph showing the results of FIG. 24A quantified. FIG.
FIG. 24C is a graph showing the results of FIG. 24A quantified. FIG.
Figure 24d is a series of photographs showing control or EZH2 inhibitor treated (DNZep treated) anomalous teratocarcinoma (ATRT) at 3, 5, 8 and 10 days after treatment. Inhibition of EZH2 inhibits ATRT cell self-renewal.
FIG. 24E is a graph showing the results of FIG. 24D quantified. FIG.
25A is a pair of graphs showing the survival fraction of untreated or DZNEP-treated (BT-16 ATRT cell line derived) ATRT cells exposed to 2Gy radiation. Inhibition of EZH2 radiosensitizes ATRT.
25B is a pair of graphs showing the survival fraction of untreated or DZNEP treated ATRT cells (derived from UPN737 ATRT cell line, "737 &quot;) exposed to 2Gy radiation. Inhibition of EZH2 radiosensitizes ATRT.
26A is a graph showing the concentration (total cell number per milliliter) of the squamous cell line GSK-126 and EZH2 as a function of time (days) after treatment with the small molecule inhibitor. Small molecule inhibitors of EZH2 reduce mesoblast cell growth.
FIG. 26B is a graph showing the concentration (total cell number per milliliter) of the squamous cell line as a function of time (days) after treatment with small molecule inhibitor of UNC 1999, EZH2. Small molecule inhibitors of EZH2 reduce mesoblast cell growth.
FIG. 26C is a graph showing the concentration (total cell number per milliliter) of the hydroblastoma cells as a function of time (days) after the treatment with small molecule inhibitor of thalassometast (EPZ 6438), EZH2. Small molecule inhibitors of EZH2 reduce mesoblast cell growth.
FIG. 26D is a graph showing the concentration (number of total cells per milliliter) of a cell of the blastomere as a function of time (days) after treatment with GSK-126, UNC 1999 and thalassemostat (EPZ 6438). Among the small molecule inhibitors tested, thymometast has the greatest effect on the growth of squamous cell line.
Figure 27 is a pair of schematic illustrations depicting the relative selectivity of thymometast for EZH2.
28A is a schematic diagram showing a method in which primary ameloblastoma cell growth is evaluated in vitro.
Figure 28b shows the relative abundance ratio (percentage of cells) of untreated or basal metastat (EPZ 6438) treated primary malignant blastoma cells in various cell cycle stages (Go / G1, Go / G1, S or G2 / FIG. The slice culture of the hydroblastoma was newly isolated from the 5 year old subject. The flaked culture was treated for 4 days with thalassemostat, then divided and analyzed by flow cytometry. Tazetate treatment reduces the growth of primary extracellular &lt; RTI ID = 0.0 &gt;&lt; / RTI &gt;
Figure 28c is a graph showing BrdU expression of the cells analyzed in Figure 28b. Tazetate treatment reduces the growth of primary extracellular &lt; RTI ID = 0.0 &gt;&lt; / RTI &gt;
29A is a graph showing the survival percentage of vehicle or in vivo other methotart (EPZ 6438) -treated ATRT cells as a function of time after treatment (days). Tazemethostat reduces ATRT in vivo.
29B is a photograph of the Western blotting results showing the relative amounts of H2K27me3 and H3 in the vehicle or the thera methotest (EPZ 6438) -treated ATRT cells of FIG. 29A.

The present disclosure provides a method of treating a hydroblastoma in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an enhancer (EZH2) inhibitor of the esthetic estrogen 2. The method of treatment of hydroblastoma of the present disclosure may include preventing and / or inhibiting the proliferation of hydroblastoma cells.

The present disclosure provides a method for treating or alleviating symptoms of SWI / SNF-associated cancers in a subject by administering a therapeutically effective amount of an EZH2 inhibitor to a subject in need of treatment or alleviation of symptoms of the SWI / SNF- to provide. For example, the SWI / SNF associated arm is characterized by reduced expression and / or loss of one or more components of the SWI / SNF complex or SWI / SNF complex. In a preferred embodiment, the cancer is a hydrobromide.

The hydroblastoma results from reduced expression and / or loss of one or more components of the SWI / SNF complex or SWI / SNF complex (including, but not limited to, SNF5, ATRX and ARID1A). For example, loss of function is caused by loss of function mutation resulting from point mutation, deletion and / or insertion.

For example, the subject is a subject with SNF5 deletion.

For example, the subject may include a substitution of wild-type residue lysine (K) with asparagine (N) (K688N) at amino acid position 688 of SEQ ID NO: 5 and a wild-type residue methionine (M) at amino acid position 366 of SEQ ID NO: (I) (M366I). &Lt; / RTI &gt;

For example, the subject may have a nonsense mutation (C884 *) of the wild-type residue cysteine (C884) at amino acid position 884 of SEQ ID NO: 11, a substitution of wild-type residue glutamic acid (E) E966K), nonsense mutation (Q1411 *) of wild type residue glutamine (Q) at amino acid position 1411 of SEQ ID NO: 11, frame shift mutation (F1720fs) at amino acid position 1720 of wild type residue phenylalanine (F) (G1847fs) behind the wild type residue glycine (G), the amino acid position 1874 of SEQ ID NO: 11, the frame shift mutation (C1874fs) at the wild type residue cysteine (C) Substitution of wild type residue aspartic acid (D) with glutamic acid (E) (D1957E), nonsense mutation (Q1430 *) of wild type residue glutamine (Q) at amino acid position 1430 of SEQ ID NO: 11, (G1255E) of wild-type residue glycine (G) at amino acid position 1255 (G1255E), amino acid position 284 of SEQ ID NO: 11 (G284fs), the nonsense mutation (R1722 *) of the wild type residue arginine (R) at amino acid position 1722 of SEQ ID NO: 11, the amino acid position 274 of SEQ ID NO: 11, the wild type residue methionine (G1847fs) in amino acid position 1847, wild-type residue G (G1847fs), amino acid position 559 in SEQ ID NO: 11, and the framework shift mutation in wild type residue P (P559fs), the nonsense mutation (R1276 *) of the wild type residue arginine (R) at position 1276 of SEQ ID NO: 11, the amino acid position 2176 of SEQ ID NO: 11, the wild type residue glutamine (Q) (H203fs) at amino acid position 203 of SEQ ID NO: 11, frame shift mutation (H203fs) at wild-type residue histidine (H), amino acid position 591 of SEQ ID NO: 11 frame shift mutation at wild type residue alanine Nonsense mutation (Q1322 *) of wild type residue glutamine (Q) at amino acid position 1322 of SEQ ID NO: 11, nonsense mutation (S2264 *) of wild type residue serine (S) at amino acid position 2264 of SEQ ID NO: 11, (Q586 *) of wild-type residue glutamine (Q) at amino acid position 586 of SEQ ID NO: 11, frame shift mutation (Q548fs) at wild-type residue glutamine (Q) at amino acid position 548 of SEQ ID NO: 11, 11 of the amino acid position 756 of the wild type residue asparagine (N) (N756fs).

The present disclosure also provides a method of identifying a subject, comprising: (a) determining the level of expression in a sample obtained from a subject, the at least one gene selected from the group consisting of a neural differentiation gene, a cell cycle arresting gene and a tumor suppressor gene; (b) selecting subjects exhibiting reduced expression levels of at least one gene in step (a); And (c) administering an effective amount of an EZH2 inhibitor to the subject selected in step (b) to treat or ameliorate the symptoms of cancer in said subject, Provides a method of treating or alleviating symptoms of this SWI / SNF-associated cancer in a subject. In a preferred embodiment, the cancer is a hydrobromide.

(A) determining the level of expression of at least one gene selected from the group consisting of a hedgehog pathway gene, a myc pathway gene and a histone methyltransferase gene in a sample obtained from a subject; (b) selecting subjects having increased expression levels of at least one gene in step (a); And (c) administering an effective amount of an EZH2 inhibitor to the subject selected in step (b) to treat or ameliorate the symptoms of cancer in said subject, Additional methods of treating or alleviating symptoms of this SWI / SNF-associated cancer in the subject are provided. In a preferred embodiment, the cancer is a hydrobromide.

For example, the neuronal differentiation gene is CD133, DOCK4 or PTPRK.

For example, the cell cycle arresting gene is CKDN1A or CDKN2A.

For example, the tumor suppressor gene is BIN1.

For example, the hedgehog pathway gene is GLI1 or PTCH1.

For example, the myc path gene is MYC.

For example, the histone methyltransferase gene is EZH2.

The present disclosure also provides methods of inducing neuronal differentiation, cell cycle arrest, or tumor suppression by contacting a cell with an EZH2 inhibitor. The EZH2 inhibitor may be present in an amount sufficient to increase expression of at least one gene selected from the group consisting of CD133, DOCK4, PTPRK, CKDN1A, CDKN2A and BIN1.

The present disclosure also provides a method of inhibiting hedgehog signaling by contacting a cell with an EZH2 inhibitor. The EZH2 inhibitor may be present in an amount sufficient to reduce the expression of GLI1 and / or PTCH1.

The present disclosure also provides a method of contacting a cell with an EZH2 inhibitor to induce gene expression. The EZH2 inhibitor may be present in an amount sufficient to induce neuronal differentiation, cell cycle arrest, and / or tumor suppression. For example, the gene may be CD133, DOCK4, PTPRK, CKDN1A, CKDN2A or BIN1.

The present disclosure also provides a method of inhibiting gene expression by contacting an EZH2 inhibitor with a cell. The EZH2 inhibitor is present in an amount sufficient to inhibit hedgehog signaling. For example, the gene may be GLI1 or PTCH1.

For example, cells may lose function of SNF5, ARID1A, ATRX, and / or SWI / SNF complex components.

For example, loss of function is caused by deletion of SNF5.

For example, the cell is a cancer cell. Preferably, the cancer is a mesoblast.

For example, an EZH2 inhibitor

(Thamethostat), or a pharmaceutically acceptable salt thereof.

For example, an EZH2 inhibitor

Their stereoisomers, their pharmaceutically acceptable salts and / or solvates.

For example, an EZH2 inhibitor

Or a pharmaceutically acceptable salt thereof.

For example, an EZH2 inhibitor

,

,

Stereoisomers, pharmaceutically acceptable salts and / or solvates thereof.

For example, an EZH2 inhibitor

,

,

Stereoisomers, pharmaceutically acceptable salts and / or solvates thereof.

For example, an EZH2 inhibitor

,

,

Their stereoisomers, their pharmaceutically acceptable salts and / or solvates.

The human nucleic acid and amino acid sequences of the SWI / SNF complex components have been described previously. For example, gene bank accession numbers NP_003064.2, NM_003073.3, NP_001007469.1 and NM_001007468.1 (in the case of SNF5), gene bank accession numbers NM_000489.3 and NP_000480, respectively, each of which is incorporated herein by reference .2, NM_138270.2 and NP_612114.1 (for ATRX), gene bank accession numbers NP_006006.3, NM_006015.4, NP_624361.1 and NM_139135.2 (for ARID1A).

The extent of hSNF5 somatic mutation in humans is also described in the references cited therein, such as Sevenet et al. , &Lt; / RTI &gt; Human Molecular Genetics, 8: 2359-2368, 1999).

Subjects in need of treatment of the present disclosure may have decreased expression, haploid insufficiency and / or SNF5 dysfunction of SNF5. For example, the subject may comprise deletion of SNF5 in a nucleic acid sequence encoding a SNF5 polypeptide or SNF5 polypeptide.

Subjects in need of treatment of this disclosure may exhibit reduced expression, haploid deficiency, and / or ATRX dysfunction of ATRX. For example, the subject may include a substitution of wild-type residue lysine (K) with asparagine (N) (K688N) at amino acid position 688 of SEQ ID NO: 5 and a wild-type residue methionine (M) at amino acid position 366 of SEQ ID NO: (I) &lt; / RTI &gt; (M366I).

Subjects requiring treatment of the present disclosure may have reduced expression, haploid deficiency and / or loss of ARID1A of ARID1A. For example, the subject may have a nonsense mutation (C884 *) of the wild-type residue cysteine (C884) at amino acid position 884 of SEQ ID NO: 11, a substitution of wild-type residue glutamic acid (E) E966K), nonsense mutation (Q1411 *) of wild type residue glutamine (Q) at amino acid position 1411 of SEQ ID NO: 11, frame shift mutation (F1720fs) at amino acid position 1720 of wild type residue phenylalanine (F) (G1847fs) behind the wild type residue glycine (G), the amino acid position 1874 of SEQ ID NO: 11, the frame shift mutation (C1874fs) at the wild type residue cysteine (C) Substitution of wild type residue aspartic acid (D) with glutamic acid (E) (D1957E), nonsense mutation (Q1430 *) of wild type residue glutamine (Q) at amino acid position 1430 of SEQ ID NO: 11, (G1255E) of wild-type residue glycine (G) at amino acid position 1255 (G1255E), amino acid position 284 of SEQ ID NO: 11 (G284fs), the nonsense mutation (R1722 *) of the wild type residue arginine (R) at amino acid position 1722 of SEQ ID NO: 11, the amino acid position 274 of SEQ ID NO: 11, the wild type residue methionine (G1847fs) in amino acid position 1847, wild-type residue G (G1847fs), amino acid position 559 in SEQ ID NO: 11, and the framework shift mutation in wild type residue P (P559fs), the nonsense mutation (R1276 *) of the wild type residue arginine (R) at position 1276 of SEQ ID NO: 11, the amino acid position 2176 of SEQ ID NO: 11, the wild type residue glutamine (Q) (H203fs) at amino acid position 203 of SEQ ID NO: 11, frame shift mutation (H203fs) at wild-type residue histidine (H), amino acid position 591 of SEQ ID NO: 11 frame shift mutation at wild type residue alanine Nonsense mutation (Q1322 *) of wild type residue glutamine (Q) at amino acid position 1322 of SEQ ID NO: 11, nonsense mutation (S2264 *) of wild type residue serine (S) at amino acid position 2264 of SEQ ID NO: 11, (Q586 *) of wild-type residue glutamine (Q) at amino acid position 586 of SEQ ID NO: 11, frame shift mutation (Q548fs) at wild-type residue glutamine (Q) at amino acid position 548 of SEQ ID NO: 11, 11 of the amino acid position 756 of the wild type residue asparagine (N) (N756fs). As used herein, "*" refers to the termination codon. As used herein, "fs" refers to frame movement.

As used herein, the term "inducing neuronal differentiation " refers to inducing the development of a cell into a cell.

According to the methods of the present disclosure, a "normal" cell can be used as a comparison criterion for expression and / or function of one or more characteristics of a cancer cell, for example, SNF5, ATRX and / or ARID1A. As used herein, "normal cell" is a cell that can not be classified as part of a "cell proliferative disorder". Normal cells do not have uncontrolled or abnormal growth, or both, that can lead to the development of an unwanted condition or disease. Preferably normal cells express an amount of EZH2 comparable to cancer cells. Preferably, the normal cells contain a wild-type sequence for the SNF5, ATRX and / or ARID1A genes and express the mutant-free SNF5, ATRX and / or ARID1A transcripts, with no mutations and all functions maintained at normal activity levels SNF5, ATRX and / or ARID1A protein.

As used herein, "contacting a cell" refers to a condition in which the compound or other composition of matter is brought into direct contact with the cell or sufficiently close to induce a desired biological effect in the cell.

As used herein, "treating" or "treating" describes the management and care of a subject to inhibit any disease, condition or disease and includes administration of an EZH2 inhibitor of this disclosure or a pharmaceutically acceptable salt thereof Or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, to alleviate the symptoms or complications of the cancer, or to remove the cancer.

As used herein, "alleviating" is intended to describe a process of reducing the severity of a symptom or symptom of cancer. The important point is that symptoms and symptoms can be alleviated without being eliminated. In a preferred embodiment, administration of the pharmaceutical compositions of the present disclosure results in the elimination of signs or symptoms, but it is not necessary to eliminate them. Effective doses are expected to reduce the severity of symptoms or symptoms. For example, a symptom or symptom of a disease, such as cancer, that can occur in multiple lesions is mitigated if the severity of the cancer is reduced in at least one of the multiple lesions.

As used herein, the term "severity" is intended to describe the potential for cancer to be transformed from a pre-cancerous or benign state to a malignant state. Alternatively or additionally, the severity may be determined by, for example, following the TNM system (approved by the International Cancer Coalition (UICC) and United States United States Common Cancer Committee (AJCC)), or by other methods known in the art It is intended to describe the cancer stage. The stage of cancer refers to the degree or severity of cancer, based on factors such as primary tumor location, tumor size, number of tumors, and lymph node involvement (cancer spread to the lymph nodes). Alternatively or additionally, the severity is intended to describe the tumor grade by methods known in the art (see National Cancer Institute, www.cancer.gov). Tumor grading is a system used to classify cancer cells in terms of how abnormal the cancer cells appear on the microscope and how quickly the tumor grows and spreads. When the tumor grade is determined, a number of factors are considered, including the structure of the cell and the growth pattern. The specific factors used to determine tumor grade depend on each type of cancer. Severity also describes a histological grade, also referred to as 'differentiation' (see the National Cancer Institute, www.cancer.gov), which tells how many of the tumor cells resemble normal cells of the same type of tissue. Furthermore, severity describes the size and shape of nuclear nuclei in tumor cells and the nuclear grade, which is the percentage of tumor cells that are dividing (see National Cancer Institute, www.cancer.gov).

In another aspect of the disclosure, severity describes the degree to which a tumor secretes growth factors, degrades extracellular matrix, forms vascular tissue, loses adherence to the juxtaposition, or is metastatic. Also, severity describes the number of lesions where primary tumors metastasize. Finally, severity involves difficulties in the treatment of tumors observed in various types and lesions. For example, cancers that are not operable, cancers that have more access to multiple body systems (hematologic and immunological tumors), and those with the most resistance to traditional therapies are considered the most serious. In such situations, it may be desirable to extend the life expectancy of the subject and / or to reduce pain, to decrease the proportion of cancerous cells or to limit the cells to any system, and to the stage / Improvement in grade / nuclear differentiation is considered to alleviate signs or symptoms of cancer.

The term "symptom" as used herein is defined as an indication of a disease, condition, injury or any phenomenon which is undesirable in the body. Symptoms are not felt or perceived by individuals experiencing the symptoms, and can not be easily perceived by others. The person at this time is defined as a person who is not a health care professional.

The term "indication" as used herein is also defined as an indication of any phenomenon that is not desirable to occur in the body. However, the indications are defined as those that can be observed by doctors, nurses or other health care professionals.

Cancer is a group of diseases that can cause almost any sign or symptom. Signs and symptoms will vary depending on where the cancer has occurred, the size of the cancer, and how much it affects nearby organs or structures. If the cancer spreads (transitions), symptoms can appear in different parts of the body.

As the cancer grows, the cancer begins to push out nearby organs, blood vessels, and nerves. This pressure causes some signs and symptoms of cancer. Cancer can occur at a location where it does not cause any symptoms until the cancer has grown fairly large.

Cancer may also cause symptoms such as, for example, fever, fatigue or weight loss. This may be because cancer cells release most of their body energy supply or release substances that alter their body metabolism. Or, cancer can induce the immune system to respond in a way that causes these symptoms. While the above listed signs and symptoms are the more common signs and symptoms observed in cancer, there are many other signs and symptoms that are less common and not listed here. However, both the signs and symptoms of cancer recognized by the art are considered and are included in the present disclosure.

Cancer treatment can result in a decrease in tumor size. A decrease in tumor size may also be referred to as "tumor regression ". Preferably, the tumor size after treatment according to the method of the present disclosure is reduced by at least 5% relative to the size of the pre-treatment itself; More preferably the tumor size is reduced by more than 10%; More preferably by at least 20%; More preferably by at least 30%; More preferably by at least 40%; Even more preferably by at least 50%; Most preferably by at least 75%. The size of the tumor can be measured by any reproducible measurement method. Tumor size can be measured as the diameter of the tumor.

Cancer treatment can result in a decrease in tumor volume. Preferably, the tumor volume after treatment according to the method of the present disclosure is reduced by at least 5% relative to the pre-treatment tumor size; More preferably the tumor volume is reduced by at least 10%; More preferably by at least 20%; More preferably by at least 30%; More preferably by at least 40%; Even more preferably by at least 50%; Most preferably by at least 75%. Tumor volume can be measured by any reproducible measurement method.

Cancer treatment can result in a decrease in the number of tumors. Preferably, the number of tumors after treatment decreases by at least 5% relative to the number of tumors before treatment; More preferably, the number of tumors is reduced by more than 10%; More preferably by at least 20%; More preferably by at least 30%; More preferably by at least 40%; Even more preferably by at least 50%; Most preferably by at least 75%. The number of tumors can be measured by any reproducible measurement method. Tumor counts can be measured by counting tumors that can be identified in or can be identified when expanded to a specific magnification. Preferably, the specific magnification is 2 X, 3 X, 4 X, 5 X, 10 X, or 50 X.

Cancer treatment can result in a decrease in the number of metastatic lesions in other tissues or organs away from the primary tumor site. Preferably, after treatment by the method of this disclosure, the metastatic lesion count is reduced by at least 5% relative to the pre-treatment metastatic lesion count; More preferably the number of metastatic lesions is reduced by more than 10%; More preferably by at least 20%; More preferably by at least 30%; More preferably by at least 40%; Even more preferably by at least 50%; Most preferably by at least 75%. The number of metastatic lesions can be measured by any reproducible measurement method. The number of metastatic lesions can be determined by counting the number of metastatic lesions that can be identified within or can be identified when expanded to a specific magnification. Preferably, the specific magnification is 2 X, 3 X, 4 X, 5 X, 10 X, or 50 X.

The effective amount of an EZH2 inhibitor of this disclosure or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof is not significantly cytotoxic to normal cells. For example, if the EZH2 inhibitor of the present disclosure is administered in a therapeutically effective amount to induce cell death in excess of 10% of normal cells, the therapeutically effective amount of the EZH2 inhibitor of this disclosure will be significantly It is not cytotoxic. The therapeutically effective amount of the EZH2 inhibitor of the present disclosure does not significantly affect the viability of normal cells when the compound is administered in a therapeutically effective amount to induce cell death in excess of 10% of normal cells.

Contacting cells with an EZH2 inhibitor of this disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, may selectively inhibit EZH2 activity in a cancer cell. Administration of an EZH2 inhibitor of this disclosure, or a pharmaceutically acceptable salt, prodrug, metabolite, polymorph, or solvate thereof, to a subject in need of the EZH2 inhibitor of the present disclosure, Can be selectively suppressed.

Male blastoma

Mesoblastoma is a fast-growing, aggressive, high-grade brain tumor. Regardless of the subtype, hydroblastoma always occurs in the cerebellum of the brain, and more specifically in the larynx of the cerebellum. The cerebellum controls balance and other complex motor functions.

Mesoblastomas rarely spread beyond the central nervous system (CNS) (ie the brain and spinal cord); Metastatic hydroblastoma can spread to bone and bone marrow. Mesenchymal cells arise from immature cells of the cerebellum that produce and replace the cerebellum cells with frequent cleavage under normal conditions.

Mesenchymal tumors are relatively rare, accounting for less than 2% of total primary brain tumors and less than 18% of total pediatric brain tumors. Over 70% of all childhood bladder tumors are diagnosed in children younger than 10 years. Mesoblastoma can also occur in adults, which, when found, are most common in adults between 20 and 44 years of age. Male blastomas occur more frequently in males than in females.

Subtypes of hydroblastoma include, but are not limited to, traditional hydroblastoma, fibrotic nodular hydroblastoma, large or retroformed hydroblastoma, hydroblastoma with neuroblastoma or neuronal differentiation, hydroblastoma with glial differentiation, Including, but not limited to, &lt; RTI ID = 0.0 &gt; glioblastoma. &Lt; / RTI &gt; The term "hydrobromide" as used in this disclosure may include all subtypes of this cancer. Mesoblastoma can also be referred to as cerebellar primitive neuroectodermal tumors (PNET).

Symptoms of hydroblastoma include symptoms of behavior change, appetite change, and increased brain pressure (eg, headache, nausea, vomiting and sleepiness, as well as problems with coordination (eg, dull behavior, difficulty in writing, Visual problems)), but is not limited thereto. Abnormal eye movements may also occur. When the cancer is spread to the spinal cord, the symptoms may include back pain, gait abnormality, and / or bladder and bowel function control abnormalities.

Mesoblastoma is often treated as a first-line treatment with surgery, followed by radiotherapy and / or chemotherapy. The subject of the present disclosure in need of a treatment in which an EZH2 inhibitor is used and preferably a therapy in which thalassemostat is used can be treated with an EZH2 inhibitor in conjunction with surgery, radiation therapy, and / or chemotherapy . Subjects of this disclosure who require treatment with an EZH2 inhibitor and preferably with a treatment with thalassemostat may be treated prior to the therapy in which the EZH2 inhibitor of the present disclosure is used with surgery, Chemotherapy may have proceeded. Subjects of this disclosure who require treatment with an EZH2 inhibitor and preferably with a treatment with thalassemostat may be treated prior to the therapy in which the EZH2 inhibitor of the present disclosure is used with surgery, May have undergone chemotherapy and have not experienced the effects of surgery, radiation therapy, and / or chemotherapy. An EZH2 inhibitor of the present disclosure, including but not limited to thalassemetast, may be used as a first line therapy before surgery, radiotherapy, and / or chemotherapy is recommended or performed on the subject.

EZH2 inhibitor

The EZH2 inhibitor of the present disclosure is selected from the group consisting of thalassemostat (EPZ-6438 or Compound A)

Or a pharmaceutically acceptable salt thereof.

Thagemethostat is also described in U.S. Patent Nos. 8,410,088, 8,765,732 and 9,090,562, the contents of each of which are incorporated herein by reference in their entirety.

The thalassemtostat or a pharmaceutically acceptable salt thereof as described herein is effective in targeting both WT EZH2 and mutant EZH2. Tazetimethastine is orally administered and is available in vivo and is highly selective for EZH2 when compared to other histone methyltransferases (i. E. The selectivity is over 20,000 times according to Ki). Importantly, thaemetostat exhibits targeted methylmark inhibition resulting in the death of genetically defined cancer cells in vitro. The animal model also showed sustained in vivo efficacy after inhibition of the target methylmark. Clinical trial results described herein also demonstrate the safety and efficacy of thymometastine.

In one embodiment, tazetate or a pharmaceutically acceptable salt thereof is administered to a subject in an amount of from about 100 mg to about 3200 mg per day, for example from about 100 mg BID to about 1600 mg BID (For example, 100 mg BID, 200 mg BID, 400 mg BID, 800 mg BID or 1600 mg BID). In one embodiment, the dose is 800 mg BID.

The EZH2 inhibitor of the present disclosure

Or stereoisomers thereof, or pharmaceutically acceptable salts and solvates thereof, or may consist essentially of, or consist of, these.

The EZH2 inhibitor of the present disclosure comprises Compound E:

Or a pharmaceutically acceptable salt thereof, or may consist essentially of, or consist of, any of these.

The EZH2 inhibitor of the present disclosure has the formula:

GSK-126, a stereoisomer thereof, a pharmaceutically acceptable salt or solvate thereof, or may consist essentially of, or consist of, these.

The EZH2 inhibitor of the present disclosure comprises Compound F:

,

,

Or stereoisomers thereof, or pharmaceutically acceptable salts and solvates thereof, or may consist essentially of, or consist of, these.

The EZH2 inhibitor of the present disclosure comprises compounds Ga to Gc:

Or a stereoisomer, pharmaceutically acceptable salt or solvate thereof, or may consist essentially of, or consist of, any of the foregoing.

EZH2 inhibitors of the present disclosure may comprise, consist essentially of, or consist of CPI-1205 or GSK343.

Additional suitable EZH2 inhibitors will be apparent to those skilled in the art. In some embodiments of the strategies, treatment modalities, methods, combinations, and compositions provided herein, an EZH2 inhibitor is a compound that describes GSK-126 in other compounds, each of which is incorporated herein by reference, U.S. Patent No. 8,536,179 (corresponding to 2011/140324).

In some embodiments of the strategies, treatment modalities, methods, combinations, and compositions provided herein, the EZH2 inhibitors are described in PCT / US2014 / 015706, PCT / US2014 / 015706 published as WO 2014/124418, each of which is incorporated herein by reference in its entirety PCT / US2013 / 025639, published as 2013/120104, and EZH2 inhibitors as described in US 14 / 839,273, published as US 2015/0368229.

In one embodiment, the compounds disclosed herein are the compounds themselves, i.e. free bases or "naked" molecules. In other embodiments, the compound is a salt thereof in its own right, for example the mono-HCl salt or tri-HCl salt, mono-HBr salt or tri-HBr salt of the excreted molecule.

The compounds disclosed herein containing nitrogen can be converted to the N-oxide by treatment with an oxidizing agent (e. G., 3-chloroperoxybenzoic acid ( m CPBA) and / or hydrogen peroxide) Other compounds suitable for the methods can be produced. Therefore, when allowed by valency and structure, all of the nitrogen-containing compound shown in the claimed invention are the compounds as shown in the present ^invention both its N- oxide derivatives, (N → O or N ⁺ -O, which may be expressed as) And the like. In yet another instance, the nitrogen of the compounds disclosed herein may be converted into an N-hydroxy or N-alkoxy compound. For example, the N-hydroxy compound can be prepared by oxidation of the parent amine with an oxidizing agent such as m- CPBA. When allowed by valency and structure, all nitrogen-containing compounds depicted and claimed in the present invention are also encompassed by the compounds shown in the present invention and their N-hydroxy derivatives (i.e., N-OH) and N-alkoxy derivatives OR, where R is substituted or unsubstituted C ₁ -C ₆ alkyl, C ₁ -C ₆ alkenyl, C ₁ -C ₆ alkynyl, 3 to 14 membered carbocycle or 3 to 14 membered heterocycle) Both are considered to include.

"Isomerism phenomenon" means that a compound has the same molecular formula but has its own atomic bonding order or its own atomic space arrangement. Isomers that differ in their atomic spatial arrangements are referred to as "stereoisomers ". Stereoisomers that are not mirror images of one another are referred to as "diastereoisomers ", and stereoisomers that are asymmetric mirror images of one another are termed" enantiomers "or sometimes optical isomers. A mixture containing equal amounts of each enantiomeric form that is opposite in chirality is referred to as a "racemic mixture ".

The carbon atom bonded to the four nonidentical substituents is referred to as the "chiral center ".

"Chiral isomer" means a compound having at least one chiral center. Compounds having more than one chiral center may be present as each diastereomer or may be present as a mixture of diastereomers referred to as a "diastereomer mixture &quot;. When one chiral center is present, the stereoisomer can be characterized by the absolute configuration (R or S) of this chiral center. Absolute placement refers to the spatial arrangement of substituents attached to the chiral center. Substituents attached to the chiral center under consideration are ranked according to the ordering rules of Khan, Ingold and Preloglog (Cahn et al ., Angew. Chem. Inter. , 1966, 5, 385; et al, Angew Chem 1966, 78 , 413;... Cahn and Ingold, J. Chem Soc 1951 (London), 612;.. Cahn et al, Experientia 1956, 12, 81;.. Cahn, J. Chem Educ . 1964, 41, 116).

"Geometric isomer" means a diastereomer in which the presence of itself is a double bond or a bond rotation centered on a cycloalkyl linker (e.g., 1,3-cyclobutyl). These arrangements can be further divided into the prefixes 'cis' and 'trans', or 'Z' and 'E', which indicate that the groups are present on the same or opposite side of intramolecular double bonds according to the C- And is distinguished by its own name.

It is to be understood that the compounds disclosed herein may be designated as different chiral isomers or geometric isomers. It is also to be understood that all isomeric forms are intended to be encompassed within the scope of this disclosure when the compounds have chiral isomeric forms or geometric isomeric forms, and that the naming of the compounds does not exclude any isomeric forms.

As well as the structures discussed in this disclosure and other compounds include all its atropic isomers. "Atropic isomers" are a class of stereoisomers in which the atoms of two isomers are spatially arranged differently. The atropic isomer is itself responsible for the limited rotation caused by interfering with the rotation of giants around the central bond. Although such atropisomers are usually present as mixtures, the development of modern chromatographic techniques has made it possible to separate two mixtures of atropic isomers when selected.

A "tautomer" is one of two or more structural isomers present in equilibrium and is readily converted from one isomer form to another isomer form. This transition results in a regular shift of the hydrogen atom with the switch of adjacent conjugated double bonds. Tautomers exist as a mixture of tautomeric sets in solution. The chemical equilibrium of the tautomers in the tautomerizable solution will be achieved. The exact ratio of tautomers depends on several factors including temperature, solvent and pH. The concept of tautomers that can be interconverted by tautomerism is referred to as a tautomerism phenomenon.

Two of the various types of feathering phenomena possible are generally observed. In keto-enol tropism, simultaneous transport of electrons and hydrogen atoms occurs. The ring-chain tautomerism occurs due to the aldehyde group (-CHO) in the same sugar chain molecule, which reacts with one of the hydroxyl groups (-OH) in the sugar chain molecule, (Annular) as shown by &lt; / RTI &gt;

Typical tautomeric pairs include, but are not limited to, ketone-enol, amide-nitrile, lactam-lactam, amide-imidic acid (e.g. in nucleobases such as guanine, thymine and cytosine) -Enamin and enamin-enamin are mutagenic phenomena. An example of a keto-enol equilibrium is the equilibrium between pyridin-2 (1H) -one and the corresponding pyridin-2-ol, as shown below.

Pyridin-2 (1 H )-On    Pyridin-2-ol

It should be understood that the compounds disclosed herein may be represented by different tautomers. It is also to be understood that when a compound has tautomeric forms, all tautomeric forms are intended to be included within the scope of this disclosure, and that the designation of the compound does not exclude any tautomeric forms.

The compounds disclosed herein include not only the compounds themselves, but also their salts and solvates, where applicable. For example, the salt may be formed between an anion and a positively charged group (e. G., An amino group) on an aryl- or heteroaryl-substituted benzene compound. Suitable anions include, but are not limited to, chlorides, bromides, iodides, sulfates, bisulfates, sulfamates, nitrates, phosphates, citrates, methanesulfonates, trifluoroacetates, glutamates, glucuronates, glutarates, Maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate and acetate (for example, trifluoroacetate) The term " pharmaceutically acceptable anion "refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, a salt can also be formed between a cation and a negatively charged group (e.g., a carboxylate) on an aryl- or heteroaryl-substituted benzene compound. Suitable cations include sodium, potassium, magnesium, calcium and ammonium cations, such as tetramethylammonium ions. The aryl- or heteroaryl-substituted benzene compounds also include salts containing a quaternary nitrogen atom. When in salt form, the ratio of the compound to the cation or anion in the salt may be 1: 1 or any ratio other than 1: 1, for example 3: 1, 2: 1, 1: 2 or 1: .

Additionally, the salts of the compounds disclosed herein, for example, the compounds, may exist in either the hydrated form or the non-hydrated form (anhydrous form), or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates and dihydrates. Non-limiting examples of solvates include ethanol solvates, acetone solvates, and the like.

"Solvate" means a solvent addition form containing a solvent in an amount that is either stoichiometric or non-stoichiometric. Some compounds have a tendency to trap solvent molecules in crystalline solid state at a fixed molar ratio to form solvates. If the solvent is water, the solvate formed is a hydrate, and if the solvent is an alcohol, the solvate formed is an alcoholate. A hydrate is formed by a combination of one or more water molecules and one molecule of a substance that stays as H ₂ O, which is the molecular state of water itself.

As used herein, the term "analogue" refers to a structure that is structurally related to another (such as by substitution by an atom of a different element of one atom, or in the presence of a particular functional group, or by substitution by another functional group of one functional group) Refers to chemical compounds that are similar but slightly different in composition. An analog is therefore a compound whose action and form is similar or nearly identical to the reference compound, but whose structure or origin is not.

The term "derivative" as defined herein refers to a compound having a common core structure and being substituted with various groups as described herein. For example, all of the compounds represented by formula (I) are aryl- or heteroaryl-substituted benzene compounds and have formula (I) as a common core structure.

The term " living body, such as a power distribution itself, " refers to a compound produced by the exchange of atoms or groups of atoms into another group of atoms or atoms that are broadly similar. The purpose of self-replenishment, such as living organisms, is to produce new compounds with biological properties similar to those of the parent compound. The self-replenishment of living organisms can be based on physical chemistry or topology. Examples of the carboxylic acid biosynthetic and the like include, but are not limited to, acylsulfonimide, tetrazole, sulfonate and phosphonate. See, for example, Patani and LaVoie, Chem. Rev. 96, 3147-3176, 1996).

The present disclosure is intended to include all isotopes of atoms present in the compounds of the present invention. The isotopes have the same atomic number, but the mass number includes different atoms. According to a common example, and not by way of limitation, isotopes of hydrogen include tritium and deuterium, and carbon isotopes include C-13 and C-14.

Pharmaceutical formulation

The present disclosure also provides a pharmaceutical composition comprising at least one EZH2 inhibitor as described herein, in combination with at least one pharmaceutically acceptable excipient or carrier.

A "pharmaceutical composition" is a formulation containing an EZH2 inhibitor of the present disclosure in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition has the form of a bulk or unit dosage form. Unit dosage forms are any of a variety of forms including, for example, capsules, IV bags, tablets, single pumps or vials for aerosol inhalation. The amount of active ingredient (e.g., a compound disclosed herein or a salt, hydrate, solvate, or isomer thereof) in a unit dose of the composition is an effective amount, depending on the particular treatment involved. One of ordinary skill in the art will understand that, from time to time, normal variations in dosage will need to be applied depending on the age and condition of the patient. The dose will also depend on the route of administration. Various routes are considered including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapulmonary, intramedullary and intracavitary routes. Dosage forms for topical or transdermal administration of a compound of the disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers or propellants required.

The phrase "pharmaceutically acceptable " as used herein means a compound that is within the scope of sound medical judgment and suitable for use in contact with human and animal tissues without causing excessive toxicity, inflammation, allergic response or other problems or complications Quot; refers to the case where the composition, material, composition, carrier and / or dosage form is proportional to a reasonable benefit / risk ratio.

"Pharmaceutically acceptable excipient" means an excipient useful in preparing a safe and non-toxic pharmaceutical composition, generally not biologically or otherwise undesirable, and includes excipients which are pharmaceutically acceptable for use in veterinary uses and human pharmaceutical applications, . As used herein, "pharmaceutically acceptable excipient" includes both such excipients and more than one of the two excipients.

The pharmaceutical compositions of this disclosure are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral routes such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical) and transmucosal administration. Solutions or suspensions used in parenteral, intradermal or subcutaneous applications may include the following components: sterile diluents, such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol or other Other synthetic solvents; Antibiotics such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium bisulfite; Coloring agents such as ethylenediaminetetraacetic acid; Buffers, such as acetate, citrate or phosphate; And tonicity adjusting agents, for example sodium chloride or dextrose. The pH can be adjusted using an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be contained in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

The compounds or pharmaceutical compositions of the present disclosure may be administered to a subject via a number of well known methods currently used in chemotherapeutic treatment. For example, for the treatment of cancer, the compounds of the present disclosure may be injected directly into the tumor, injected into the bloodstream or body cavity, orally ingested, or patches applied and applied through the skin. The selected dose should be sufficient to constitute a valid treatment, but not so large as to cause adverse side effects that are not allowed. The condition of the disease condition (e.g., cancer, pre-cancer, etc.) and the health of the patient should preferably be monitored closely during and over a reasonable period after treatment.

The term "therapeutically effective amount" as used herein refers to the amount of an EZH2 inhibitor, composition, or pharmaceutical composition thereof effective to treat, alleviate or prevent a specified disease or condition, or to exhibit an identifiable therapeutic or suppressive effect do. The effect can be confirmed by any analytical method known in the art. The precise effective dose for the subject will depend on the weight, size and health status of the subject; The nature and extent of the condition; And the therapeutic agent or combination of agents selected for administration. The therapeutically effective amount for a given disease can be determined by routine experimentation within the skill and judgment of the clinician. In a preferred embodiment, the disease or condition to be treated is cancer, including, but not limited to, a &lt; RTI ID = 0.0 &gt;

A therapeutically effective amount for any EZH2 inhibitor of this disclosure can be estimated first, for example, in cell culture assays of neoplastic cells, or in animal models, usually rats, mice, rabbits, dogs or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information may then be used to determine useful dosing doses and routes of administration in humans. The therapeutic / prophylactic efficacy and toxicity, for example the ED ₅₀ (therapeutically effective dose at 50% of the population) and the LD ₅₀ (lethal dose at 50% of the population), can be determined by standard pharmaceutical methods in cell cultures or laboratory animals Lt; / RTI &gt; The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as a ratio, LD ₅₀ / ED ₅₀ . A pharmaceutical composition exhibiting a high therapeutic index is preferred. The dose may vary within this range depending on the dosage form employed, the patient's susceptibility and the route of administration.

The dose and administration are adjusted to provide a sufficient level of the active agent (s) and to maintain the desired effect. Factors that may be considered include the severity of the disease state, the overall health status of the subject, the age, weight and sex of the subject, diet, time and frequency of administration, drug combination (s), response susceptibility, Response. The long-acting pharmaceutical composition may be administered every 3 to 4 days, once every week or every 2 weeks, depending on the half-life and clearance rate of the particular formulation.

The pharmaceutical compositions containing the EZH2 inhibitors of this disclosure can be prepared by generally known methods such as conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, emulsifying, have. The pharmaceutical compositions may be formulated in conventional manner using one or more pharmaceutically acceptable carriers, including excipients and / or auxiliaries, which facilitate the processing of the active compounds into preparations which can be used pharmaceutically, have. Of course, the appropriate formulation will depend on the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions and sterile aqueous solutions (if aqueous) or dispersions. Suitable carriers for intravenous administration include physiological saline, mucilage, Cremophor ?? EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it is easily injectable. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and a dispersion medium or a solvent containing a suitable mixture thereof. For example, lecithin is used, and in the case of dispersions, the required particle size is maintained and surfactants can be retained by use. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases it may be desirable to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Prolongation of the absorption time of the injectable composition may be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

The sterile injectable solution may be prepared by incorporating the required amount of the active compound into an appropriate solvent, followed by sterile filtration, together with one or a combination of the components listed above, if necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other necessary components of those listed above. In the case of sterile powders for the production of sterile injectable solutions, the preparation method is a vacuum drying method and a freeze-drying method in which powders of an optional ingredient and an active ingredient are prepared from a solution previously sterilized and filtered.

Orally administered compositions generally comprise an inert diluent or a pharmaceutically acceptable excipient. The composition may be contained in a gelatin capsule or compressed into tablets. For administration of the oral therapeutic agent, the active compound may be incorporated with excipients and used in the form of tablets, troches, or capsules. Compositions for oral administration may also be prepared using a fluid carrier to be used as an oral cleanser, The compound in this fluid carrier can be applied orally, swallowed in the mouth, gagged and then spit or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds having similar properties: binders such as microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starches or lactose; Disintegrants such as alginic acid, Primogel or corn starch; Lubricants such as magnesium stearate or Sterotes; For example, colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; Or flavoring agents, for example peppermint, methyl salicylate or orange flavor.

For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser, or sprayer containing a suitable propellant, for example a gas such as carbon dioxide.

Systemic administration may also be by light transmucosal means or transdermal means. For transmucosal administration or transdermal administration, a transdermal absorption enhancer suitable for the barrier to be permeated is used in the formulation. Such transdermal absorption accelerators are generally known in the art. For example, transdermal absorption enhancers for transmucosal administration include detergents, bile salts and fucidic acid derivatives. Transmucosal administration can be accomplished via intranasal spray or suppository. For transmucosal administration, the active compounds are formulated into ointments, salves, gels or creams as generally known in the art.

The active compound (i. E., An EZH2 inhibitor of the present disclosure) is a pharmaceutically acceptable compound that will prevent the compound from rapidly dissolving in the body as a controlled release formulation, including, for example, implants and microencapsulated delivery systems Can be made with carriers. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Methods for making such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including monoclonal antibodies to viral antigens, including liposomes targeted to infected cells) may also be used as pharmaceutically acceptable carriers. Such suspensions may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is particularly advantageous to formulate a composition for oral administration or parenteral administration in dosage unit form for ease of administration and uniformity of dosage. Dosage unit type as used herein refers to physically discrete units suitable as unit dosage forms for the subject to be treated; Each such unit, together with the required pharmaceutical carrier, contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect. The description of the dosage unit form of this disclosure is based on and is directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved.

For therapeutic applications, the dosage of the pharmaceutical composition used in accordance with the present disclosure will depend upon a variety of factors, including but not limited to the age, weight, and clinical condition of the subject being administered, Depending on the experience and judgment of the clinician or practitioner. In general, the dose should not only slow the growth of the tumor, preferably regress it, but should preferably be sufficient to cause complete regression of the cancer. An effective amount of a pharmaceutical preparation is an amount that provides an improvement that can be objectively identified as noted by the clinician or other qualified observer. For example, degeneration of a tumor in a patient can be measured with reference to the diameter of the tumor. Decrease in tumor diameter indicates regression. Degeneration is also indicated by the fact that the tumor does not recur after treatment has stopped. The term "dose-effective manner" as used herein refers to the manner in which the amount of active compound exerts the desired biological effect in a subject or cell.

The pharmaceutical composition may be contained in a container, pack or dispenser with instructions for administration.

The compounds of the present disclosure may further generate salts. All of these forms are also contemplated within the scope of the present disclosure.

As used herein, "pharmaceutically acceptable salts" refers to derivatives of the compounds of the present disclosure in which the parent compound is modified by the preparation of its acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, basic residues such as inorganic or organic acid salts of amines, acidic residues such as alkali or organic salts of carboxylic acids, and the like. Pharmaceutically acceptable salts include, for example, quaternary ammonium salts or conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts are salts of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonic acid, carbonic acid, citric acid, , Hydroxymaleic acid, hydroxycarboxylic acid, hydroxymaleic acid, hydroxymaleic acid, hydroxymaleic acid, hydroxymaleic acid, hydroxymaleic acid, hydroxymaleic acid, hydroxycarboxylic acid, But are not limited to, lactic acid, malic acid, mandelic acid, methanesulfonic acid, lead acid, nitric acid, oxalic acid, fumaric acid, pantothenic acid, phenylacetic acid, phosphoric acid, For example, glycine, alanine, phenylalanine, acetic acid, acetic acid, lactic acid, propionic acid, salicylic acid, stearic acid, secondary acetic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfuric acid, tannic acid, It includes salts derived from inorganic and organic acids are selected from such as ginin but not limited to.

Other examples of pharmaceutically acceptable salts include those derived from nucleic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, 4- chlorobenzenesulfonic acid, 2- 2-ene-1-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, muconic acid, etc. &Lt; / RTI &gt; The disclosure also relates to a process wherein the acidic proton present in the parent compound is substituted by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; Or salts formed when coordinating with organic bases, such as ethanolamine, diethanolamine, triethanolamine, tromethamine and N-methylglucamine, and the like.

It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates) of the same salt or crystalline forms (polymorphs) as defined herein.

EZH2 inhibitors of the present disclosure may also be prepared as esters, for example, as pharmaceutically acceptable esters. For example, the carboxylic acid functional groups in the compounds can be converted into their corresponding esters, such as methyl esters, ethyl esters or other esters. The alcohol groups in the compounds can also be converted into their corresponding esters, such as acetate, propionate or other esters.

EZH2 inhibitors of this disclosure may also be prepared as prodrugs, e. G., As pharmaceutically acceptable prodrugs. The terms "prodrug-drug" and "prodrug" are used interchangeably herein and refer to any compound that releases an active parent drug in vivo. Because the prodrug is known to enhance many of the desired properties of the drug (e.g., solubility, bioavailability, formulation, etc.), the compounds of the present disclosure may be delivered in the form of prodrug. Accordingly, the disclosure is intended to encompass prodrugs of the compounds claimed herein, methods of delivering them, and compositions containing them. "Prodrug" is intended to include any covalently bonded carrier that releases the active parent drug of this disclosure in vivo when such prodrug is administered to the subject. Prodrugs of this disclosure are prepared by modifying the functional groups present in the compound in such a way that the modification is cleaved or cleaved in vivo in normal operation to produce the parent compound. Prodrug can be prepared by reacting a hydroxy, amino, sulfhydryl, carboxy or carbonyl group with any of those capable of forming a free hydroxyl, free amino, free sulfhydryl, free carboxy or free carbonyl groups, Lt; / RTI &gt; group.

Examples of prodrugs include esters of hydroxy functional groups (e.g., acetate, dialkylaminoacetate, formate, phosphate, sulfate and benzoate derivatives) and carbamates (e.g., N, N-acyl derivatives of amino functional groups (e.g., N-acetyl) N-Mannich bases, N-dimethylaminocarbonyl esters of carboxylic functional groups (e.g., ethyl esters, Schiff base and enaminone, ketones and aldehyde functional oximes, acetals, ketals and enol esters, etc. [Bundegaard, H., Design of Prodrugs , p1-92, Elesevier, New York -Oxford (1985)].

The present EZH2 inhibitor, or a pharmaceutically acceptable salt, ester or prodrug thereof, may be administered orally, nasally, percutaneously, lung, inhaled, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, Intravenous and parenteral administration. In one embodiment, the compound is administered orally. One skilled in the art will recognize the advantages of a particular route of administration.

The dosage regimen in which the compound is used depends on the type, species, age, weight, sex, and medical condition of the patient; The severity of the condition being treated; The route of administration; Patient's kidney and liver function; And the specific compound or salt thereof used. The ordinary skilled practitioner or veterinarian can easily determine and prescribe the effective amount of the drug necessary to prevent, prevent or prevent the progression of the condition.

The dosage regimen may be daily administration of the compounds of this disclosure (e. G., Every 24 hours). The dosing regimen may be daily administration for consecutive days, for example at least 2, at least 3, at least 4, at least 5, at least 6 or at least 7 consecutive days. Dosage may be made more than once daily, for example, two, three or four times per day (per 24 hour period). The dosage regimen may not be administered at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 6 days after daily administration.

Formulation and administration techniques of the disclosed compounds of the present disclosure are the literature, it can be found in [Remington the Science and Practice of Pharmacy , 19 th edition, Mack Publishing Co., Easton, PA (1995)]. In one embodiment, the compounds described herein and their pharmaceutically acceptable salts are used in pharmaceutical preparations together with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents, and sterile aqueous or organic solutions. The compound will be present in such pharmaceutical compositions in an amount sufficient to provide the desired dose in the ranges described herein.

All percentages and ratios used herein are by weight unless otherwise specified.

Other features and advantages of the present disclosure become apparent from the various embodiments. The embodiments presented are illustrative of the methods and different components useful in carrying out the present disclosure. The embodiments do not limit the claimed disclosure. Those skilled in the art will recognize and use the methods and other components useful in carrying out the present disclosure based on the disclosure herein.

Example

In order that the invention disclosed herein may be more effectively understood, the following examples are provided. It is to be understood that these embodiments are for purposes of illustration only and are not to be construed as limiting the present disclosure in any way.

Example 1: Tazzemetostat reduces mature cell growth

Male blastoma cells were treated with negative control (DMSO) or one of the following concentrations: 0.5 μM, 2 μM and 6 μM thalassemostat (EPZ 6438). Total cell counts per ml of culture were counted daily for 10 days. Each of the tazetate treatments showed a significant decrease in the growth of the squamous cell cell compared to the wild type (Fig. 26C), and the effect was concentration dependent.

Compared with the efficacy of other small molecule EZH2 inhibitors, including GSK-126 and UNC 1999, thaemetostat showed excellent ability to reduce hydroblastoma cell growth (Figure 26d).

Example 2: Tazzemetostat reduces mesoblast cell growth in in vitro flaky culture medium.

The patient underwent surgery for a 5-year-old male blastoma and removed the tumor tissue fragments for examination. The hydroblastoma flakes were cultured in vitro on tissue support inserts (Fig. 28A). For 4 days, part of the flaked culture was untreated, and the other part was treated with low-concentration methotrexate (500 nM) or high-concentration methotrexate (2 μM). After the treatment period, the cells of the flaky culture broth were divided by treatment with BrdU for 4 hours and classified by flow cytometry.

FIG. 28B shows the percentage of cells in each of four cell cycle stages (G0 / G1 transfer (sub-G0 / G1), Go / G1, S or G2 / M) do. The data showed that the proportion of cells treated with thalassemostat in the G0 / G1 phase was increased and the percentage of cells treated with thalassemostat in the G2 / M phase was reduced compared to the untreated control . The data indicate that the thymometastat treatment inhibits the proliferation / growth of the mesenchymal stem cells by interfering with cell division.

Figure 28c confirms the results of Figure 28b, which demonstrates that the number of cells synthesizing DNA in the &lt; RTI ID = 0.0 &gt; thymometast-treated &lt; / RTI &gt; cells significantly decreased, as evidenced by the decreased incorporation of BrdU.

All publications and patent documents cited herein are incorporated herein by reference as if specifically and individually stated that each such publication or document is incorporated herein by reference. References to publications and patent literature are not intended as an admission that any article is related to the prior art, nor do they constitute any form of acknowledgment of the contents or date of such article. Those skilled in the art will now be able to carry out the invention described in the foregoing description by way of various embodiments and that the description and the following embodiments are for purposes of illustration only and do not limit the claims appended hereto I will recognize. Where cell lines or gene names are used, the abbreviations and names are followed by the naming of the American Type Culture Collection (ATCC) or the National Center for Biotechnology Information (NCBI) unless otherwise specified or clear from context.

The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the embodiments are to be considered in all respects as illustrative rather than limiting of the invention described herein. The scope of the invention is, therefore, defined by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims (34)

A method of treating a hydroblastoma in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an enhancer (EZH2) inhibitor of the esthetic estrogen 2. The method of claim 1, wherein the EZH2 inhibitor is

(Tazetate), or a pharmaceutically acceptable salt thereof. The method of claim 1, wherein the EZH2 inhibitor is

Or a pharmaceutically acceptable salt, solvate, solvate, stereoisomer, pharmaceutically acceptable salt and / or solvate thereof. The method of claim 1, wherein the EZH2 inhibitor is

&Lt; / RTI &gt; or a pharmaceutically acceptable salt thereof. The method of claim 1, wherein the EZH2 inhibitor is

,
Or a pharmaceutically acceptable salt, solvate, stereoisomer, pharmaceutically acceptable salt and / or solvate thereof. The method of claim 1, wherein the EZH2 inhibitor is

,
Or a pharmaceutically acceptable salt, solvate, stereoisomer, pharmaceutically acceptable salt and / or solvate thereof. The method of claim 1, wherein the EZH2 inhibitor is

Or a pharmaceutically acceptable salt, solvate, solvate, stereoisomer, pharmaceutically acceptable salt and / or solvate thereof. 8. The method according to any one of claims 1 to 7, wherein the EZH2 inhibitor is administered orally. 9. The method according to any one of claims 1 to 8, wherein the EZH2 inhibitor is formulated as an oral tablet. 10. The method according to any one of claims 1 to 9, wherein said EZH2 inhibitor is administered in a dose of from 10 mg / kg / day to 1600 mg / kg / day. 11. The method of claim 10, wherein said EZH2 inhibitor is administered at a dose of about 100 mg, about 200 mg, about 400 mg, about 800 mg, or about 1600 mg. 12. The method of claim 11, wherein the EZH2 inhibitor is administered in a dose of about 800 mg. 9. The method according to any one of claims 1 to 8, wherein the EZH2 inhibitor is formulated as an oral suspension. 9. The method according to any one of claims 1 to 8, wherein said EZH2 inhibitor is formulated for administration to brain spinal fluid (CSF). 15. The method of claim 14, wherein the EZH2 inhibitor is administered to the cerebrospinal fluid by intrathecal, intracranial, intraspinal or intranasal route. 16. The method according to any one of claims 13 to 15, wherein the EZH2 inhibitor is administered twice a day (BID) at a dose of 230 mg / m 2 to 600 mg / m 2 including the end point. 17. The method of claim 16, wherein said EZH2 inhibitor is administered twice daily (BID) at a dose of from about 230 mg / m 2 to about 305 mg / m 2, including an endpoint. 16. The method according to any one of claims 13 to 15, wherein said EZH2 inhibitor is administered twice daily (BID) in a dose of 240 mg / m &lt; 2 &gt;. 16. The method according to any one of claims 13 to 15, wherein said EZH2 inhibitor is administered twice daily (BID) in a dose of 300 mg / m &lt; 2 &gt;. 16. The method according to any one of claims 13 to 15, wherein said EZH2 inhibitor is administered at a steady state (AUC) (ACU _SS ) under a steady state after 1600 mg administered twice a day to an adult subject, RTI ID = 0.0 &gt; 60% &lt; / RTI &gt; 21. The method of claim 13 or 20, wherein the EZH2 inhibitor is administered at a dose of about 600 mg / m 2 per day. 21. The method according to claim 13 or 20, wherein the EZH2 inhibitor is administered at a dose of at least 600 mg / m 2 per day. 16. The method according to any one of claims 13 to 15, wherein the EZH2 inhibitor is administered to an adult subject in an amount of about 80% of the area under the curve (AUC) (ACU _SS ) under steady state after 800 mg twice a day, &Lt; / RTI &gt; 24. The method of claim 13 or 23 wherein said EZH2 inhibitor is administered twice daily (BID) at a dose of about 390 mg / m &lt; 2 &gt;. 24. The method of claim 13 or 23, wherein the EZH2 inhibitor is administered twice daily (BID) at a dose of at least 390 mg / m &lt; 2 &gt;. 16. The method according to any one of claims 13 to 15, wherein the EZH2 inhibitor is administered twice daily (BID) at a dose of 300 mg / m2 to 600 mg / m2.
27. The method of any one of claims 1 to 26 wherein the EZH2 inhibitor is administered twice daily (BID).
28. The method of any one of claims 1 to 27, wherein the subject is a pediatric subject.
29. The method of claim 28, wherein the subject is between 6 months and 21 years of age, including an endpoint.
30. The method of claim 29, wherein the subject is between 1 and 18 years of age, including an endpoint. 29. The method of claim 28, wherein the subject is at least 10 years of age. 29. The method of claim 28, wherein the subject is at least 5 years of age. 33. The method according to any one of claims 1 to 32, wherein the treatment comprises preventing and / or inhibiting proliferation of male pluripotent cells. A method of treating a hydroblastoma in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of thalassemostat, wherein the therapeutically effective amount is at least 300 (BID) twice daily Mg / m &lt; 2 &gt;, wherein said subject is between 6 months and 21 years of age, including the endpoint.

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